THF peroxide as a factor in generating desulphurised products from the solid-phase synthesis of phosphorothioate-modified oligonucleotides

Antisense oligonucleotides (ASOs) are generally obtained via chemical synthesis on a solid support and phosphorothioate (PS) modification with a phosphate backbone to increase their in vivo stability and activity. However, desulphurised products, in which PS is partially replaced by phosphodiesters, are generally formed during the chemical synthesis of ASO and are difficult to separate from the desired PS-modified ASO by chromatography. Therefore, revealing the unknown factors that cause the formation of desulphurised products and proposing methods to inhibit their formation are highly desirable. In this study, it was found that peroxides in THF, which is used as a solvent for the acetyl capping agent, oxidise phosphite triesters to produce desulphurisation products. The use of THF with antioxidants effectively suppresses the oxidation caused by THF peroxides. Moreover, THF peroxide was found to oxidise phosphoramidites, which are the building blocks of oligonucleotide chemical syntheses, indicating that caution should be taken with the organic solvents used during the synthesis and purification of phosphoramidites.

Fig. S3 HPLC charts of crude PS-T5-mer using solvent with adjusted peroxide concentration instead of capping reagent (THF peroxide concentration is shown in Table S2, entries 1-6).
3. Impurities contained in crude PS-T5-mer synthesised using dT phosphoramidite in THF Fig. S4 HPLC charts of PS-T5-mer synthesised using solution of thymidine phosphoramidite with THF peroxide (Solvents are shown in Table 2 and Table S3).

Oxidation of thymidine-phophoramidite by THF peroxide
Thymidine-phosphoramidite was dissolved in THF (with and without BHT) and adjusted to a final concentration of 0.067 M. A portion of the reaction solution is withdrawn after 5, 30, 60, and 120 minutes, and THF was removed in vacuo.The obtained crude residue was dissolved in methanol and the oxidation ratio was evaluated by LC/MS measurement.The amount of peroxide contained in THF with BHT is approximately 10 ppm.The amount of peroxide contained in THF without BHT is approximately 1336 ppm.

Fig. S5
Fig. S5 HPLC chart of thymidine phosphoramidite after treatment with THF a) containing BHT and b) without BHT Fig. S6 Nusinersen
): Numbers in parentheses indicate the number of desulphurised PS bonds.

Fig. S10
Fig.S10LC charts of nusinersen synthesised by acetyl capping method and POSP ratios contained in crude nusinersen according to deconvolution mass intensity (Capping reagents are shown in Table4, Entries 1 and 2.)

Fig. S14
Fig. S14 LC (UV) and MS chart of crude PS-T5-mer synthesised under conditions of flowing THF and ETT instead of cholesterol capping (Table 1, entry 4).

Fig. S15
Fig. S15 LC (UV) and MS charts of MS chart of crude PS-T5-mer synthesised under conditions of flowing MeCN and ETT instead of cholesterol capping (Table 1, entry 5).

Fig. S16
Fig. S16 LC (UV) and MS charts of MS chart of crude PS-T5-mer synthesised under conditions of flowing THF (with BHT) only instead of cholesterol capping (Table 1, entry 6).

Fig. S17
Fig. S17 LC (UV) and MS charts of MS chart of crude PS-T5-mer synthesised under conditions of flowing THF (without BHT) instead of cholesterol capping (Table 1, entry 7).

Table S1
Determination of peroxide concentration in each solvent S42.

Table S2
Peroxide concentration in each solvent and POSP ratio in the crude PS-T5-mer